Deletions of 11q22.3-q25 are associated with atypical lung carcinoids and poor clinical outcome.

Carcinoids are slow-growing neuroendocrine tumors that, in the lung, can be subclassified as typical (TC) or atypical (AC). To identify genetic alterations that improve the prediction of prognosis, we investigated 34 carcinoid tumors of the lung (18 TCs, 15 ACs, and 1 unclassified) by using array comparative genomic hybridization (array CGH) on 3700 genomic bacterial artificial chromosome arrays (resolution ≤1 Mb). When comparing ACs with TCs, the data revealed: i) a significant difference in the average number of chromosome arms altered (9.6 versus 4.2, respectively; P = 0.036), with one subgroup of five ACs having more than 15 chromosome arms altered; ii) chromosomal changes in 30% of ACs or more with additions at 9q (≥1 Mb) and losses at 1p, 2q, 10q, and 11q; and iii) 11q deletions in 8 of 15 ACs versus 1 of 18 TCs (P = 0.004), which was confirmed via fluorescence in situ hybridization. The four critical regions of interest in 45% ACs or more comprised 11q14.1, 11q22.1-q22.3, 11q22.3-q23.2, and 11q24.2-q25, all telomeric of MEN1 at 11q13. Results were correlated with patient clinical data and long-term follow-up. Thus, there is a strong association of 11q22.3-q25 loss with poorer prognosis, alone or in combination with absence of 9q34.11 alterations (P = 0.0022 and P = 0.00026, respectively).

Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability.

AIMS: To test the hypothesis that the published morphological criteria permit reliable segregation of small cell carcinoma of the lung (SCLC) and large cell neuroendocrine carcinoma (LCNEC) cases by determining the interobserver variation. METHODS AND RESULTS: One hundred and seventy cases of SCLC, LCNEC and cases diagnosed as neuroendocrine lung carcinoma before LCNEC had been established as a diagnostic category were retrieved from the archives of the assessor's institutes. A representative haematoxylin and eosin section from each case was selected for review. Batches of cases were circulated among nine pathologists with a special interest in pulmonary pathology. Participants were asked to classify the cases histologically according to the 2004 World Health Organization (WHO) criteria. The diagnoses were collected and kappa values calculated. Unanimity of diagnosis was achieved for only 20 cases; a majority diagnosis was reached for 115 cases. In 35 cases no consensus diagnosis could be reached. There was striking variability amongst assessors in diagnosing SCLC and LCNEC. The overall level of agreement for all cases included in this study was fair (kappa=0.40). CONCLUSIONS: Using non-preselected cases, the morphological WHO criteria for diagnosing SCLC and LCNEC leave room for subjective pathological interpretation, which results in imprecise categorization of SCLC and LCNEC cases.

Differential methylation of a short CpG-rich sequence within exon 1 of TCF21 gene: a promising cancer biomarker assay.

Detection of cancer cells at early stages could potentially increase survival rates in cancer patients. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. A recent report from our laboratory described the use of quantitative methylation-specific PCR assays for discriminating patients with lung cancer from those without lung cancer using lung biopsies as well as sputum samples. TCF21 is known to be essential for differentiation of epithelial cells adjacent to mesenchyme. Using restriction landmark genomic scanning, a recent study identified TCF21 as candidate tumor suppressor at 6q23-q24 that is epigenetically inactivated in lung and head and neck cancers. Using DNA sequencing technique, we narrowed down a short CpG-rich segment (eight specific CpG sites in the CpG island within exon 1) of the TCF21 gene, which was unmethylated in normal lung epithelial cells but predominantly methylated in lung cancer cell lines. We specifically targeted this short CpG-rich sequence and developed a quantitative methylation-specific PCR assay suitable for high-throughput analysis. We showed the usefulness of this assay in discriminating patients with lung cancer from those without lung cancer using biopsies and sputum samples. We further showed similar applications with multiple other malignancies. Our assay might have important implications in early detection and surveillance of multiple malignancies.

Application of a methylation gene panel by quantitative PCR for lung cancers.

Detection of lung cancer at early stages could potentially increase survival rates. One promising approach is the application of suitable lung cancer-specific biomarkers to specimens obtained by non-invasive methods. Thus far, clinically useful biomarkers that have high sensitivity have proven elusive. Certain genes, which are involved in cellular pathways such as signal transduction, apoptosis, cell to cell communication, cell cycles and cytokine signaling are down-regulated in cancers and may be considered as potential tumor suppressor genes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. Using quantitative real time PCR, we tested 11 genes (3-OST-2, RASSF1A, DcR1, DcR2, P16, DAPK, APC, ECAD, HCAD, SOCS1, SOCS3) for levels of methylation within their promoter sequences in non-small cell lung cancers (NSCLC), adjacent non-malignant lung tissues, in peripheral blood mononuclear cells (PBMC) from cancer free patients, in sputum of cancer patients and controls. Of all the 11 genes tested 3-OST-2 showed the highest levels of promoter methylation in tumors combined with lowest levels of promoter methylation in control tissues. 3-OST-2 followed by, RASSF1A showed increased levels of methylation with advanced tumor stage (P<0.05). Thus, quantitative analysis of 3-OST-2 and RASSF1A methylation appears to be a promising biomarker assay for NSCLC and should be further explored in a clinical study. Our preliminary data on the analysis of sputum DNA specimens from cancer patients further support these observations.

A prospective study of the timing and cost-effectiveness of bronchial washing during bronchoscopy for pulmonary malignant tumors.

STUDY OBJECTIVES: The value of obtaining washings during fiberoptic bronchoscopy in the workup of lung cancer is controversial. Moreover, the optimal timing of washing relative to biopsy and brushing is not known. In this study, the diagnostic yields of washings before and after biopsy and brushings were compared. The different diagnostic strategies were assessed in terms of yield and costs. DESIGN: A prospective study performed from 2001 to 2003 in a secondary care medical center. MEASUREMENTS AND RESULTS: Two hundred twenty-one patients underwent flexible bronchoscopy, and the diagnostic yield of washings before biopsy and brushing (strategy I) and after biopsy and brushing (strategy II) specimens were assessed. Using the known probabilities and costs for various bronchoscopic procedures, the expected utility of a number of diagnostic strategies was estimated. Patients (147 men and 74 women) were included in the study in whom a definite cytologic or histologic diagnosis of pulmonary malignancy had been made. The diagnostic yield of strategy I was 72% for visible tumors and 36% for nonvisible tumors. For strategy II, the diagnostic yield was 74% for visible tumors and in 42% for nonvisible tumors. The comparison of strategies I and II for both visible and nonvisible tumors revealed that 176 cases were concordant (80%); in 19 cases (9%) the cytologic analysis of washings in strategy I was positive for malignancy and negative in strategy II. In 26 cases (12%) washings in strategy II were positive and negative in strategy I (p = 0.37). An analysis of the diagnostic yield of both washings in visible tumors and nonvisible tumors showed no significant difference. In 13 patients, a diagnosis of malignancy was established only by washings (6%). Confining the laboratory investigations of washings or brush samples to those cases in which the initial findings of the biopsies are negative (the two-stage procedure) is more cost-effective than examining all biopsy, brushing, and washing specimens. In patients with visible tumors, brushing or washing in addition to biopsy is equally cost-effective; in patients with nonvisible tumors, biopsy combined with washing is the preferred option. CONCLUSIONS: No difference in the diagnostic yield could be demonstrated for washings before or after biopsies and brushings. Although the additional diagnostic yield of washing and brushing during bronchoscopy is relatively low, it is cost-effective to use these procedures in the diagnostic workup of patients who are clinically suspected of having a pulmonary malignancy.

Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH), a fatal disease of unknown etiology characterized by impaired regulation of pulmonary hemodynamics and vascular growth, is associated with low levels of pulmonary nitric oxide (NO). Based upon its critical role in mediating vasodilation and cell growth, decrease of NO has been implicated in the pathogenesis of PAH. We evaluated mechanisms for low NO and pulmonary hypertension, including NO synthases (NOS) and factors regulating NOS activity, i.e. the substrate arginine, arginase expression and activity, and endogenous inhibitors of NOS in patients with PAH and healthy controls. PAH lungs had normal NOS I-III expression, but substrate arginine levels were inversely related to pulmonary artery pressures. Activity of arginase, an enzyme that regulates NO biosynthesis through effects on arginine, was higher in PAH serum than in controls, with high-level arginase expression localized by immunostaining to pulmonary endothelial cells. Further, pulmonary artery endothelial cells derived from PAH lung had higher arginase II expression and produced lower NO than control cells in vitro. Thus, substrate availability affects NOS activity and vasodilation, implicating arginase II and alterations in arginine metabolic pathways in the pathophysiology of PAH.

Mitogen-activated protein kinase phosphatase-1 is overexpressed in non-small cell lung cancer and is an independent predictor of outcome in patients.

PURPOSE: An increase in the activity of the mitogen-activated protein kinases (MAPKs) has been correlated with a more malignant phenotype in several tumor models in vitro and in vivo. A key regulatory mechanism of the MAPKs [extracellular signal-regulated kinase (ERK); c-jun NH(2)-terminal kinase (JNK); and p38] is the dual specificity phosphatase CL100, also called MAPK phosphatase-1 (MKP-1). This study was designed to examine the involvement of CL100/MKP-1 and stress-related MAPKs in lung cancer. EXPERIMENTAL DESIGN: We assessed the expression of CL100/MKP-1 and the activation of the MAPKs in a panel of 18 human cell lines [1 primary normal bronchial epithelium, 8 non-small cell lung cancer (NSCLC), 7 small cell lung cancer (SCLC), and 2 carcinoids] and in 108 NSCLC surgical specimens. RESULTS: In the cell lines, CL100/MKP-1 expression was substantially higher in NSCLC than in SCLC. P-ERK, P-JNK, and P-p38 were activated in SCLC and NSCLC, but the degree of their activation was variable. Immunohistochemistry in NSCLC resection specimens showed high levels of CL100/MKP-1 and activation of the three MAPK compared with normal lung. In univariate analysis, no relationship was found among CL100/MKP-1 expression and P-ERK, P-JNK, or P-p38. Interestingly, high CL100/MKP-1 expression levels independently predicted improved survival in multivariate analysis. JNK activation associated with T(1-2) and early stage, whereas ERK activation correlated with late stages and higher T and N. Neither JNK nor ERK activation were independent prognostic factors when studied for patient survival. CONCLUSIONS: Our data indicate the relevance of MAPKs and CL100/MKP-1 in lung cancer and point at CL100/MKP-1 as a potential positive prognostic factor in NSCLC. Finally, our study supports the search of new molecular targets for lung cancer therapy within the MAPK signaling pathway.

Microarray as a model for quantitative visualization chemistry.

For visualization of proteins or nucleic acids, direct and indirect in situ fluorescence and absorption methods (immunohistochemistry and cytochemistry) have existed for many years. The authors describe a new experimental approach using microarray as a model to quantitatively compare both visualization methods. The spots obtained with the microarray robot had a progressive twofold decrease in concentrations and are used as objects with known amounts of DNA. Subsequent hybridization resulted in a direct fluorescence (DF) label or in hapten for indirect fluorescence (IF) and absorption. The results show that the image of the object in the IF method is larger than that in the DF method because of an edge effect, with stronger staining at the circumference. This leads to a higher plateau level and an 8- to 10-fold reduction in the detection threshold for IF compared with DF. These features are especially useful for one-color DNA-related microarray analysis, such as single nucleotide polymorphism, loss of heterozygosity, and mutation analysis, provided that the spots are not designed directly adjacent to each other, so that the edge effect is taken into account. The slope of the linear range for the IF method is much steeper than for the DF method, pointing to a narrow dynamic range in immunohistochemistry. It is noteworthy that the detection limit for absorption images after indirect immunoenzyme visualization is lower than for the DF images. The indirect immunohistochemistry semiquantitative absorption signal was at least similar compared with the DF fluorescence. In conclusion, an explanation for the difficulties experienced in quantitative immunohistochemistry is provided, and the data emphasize that in general, for daily pathology, semiquantitative patterns should suffice. Indirect labeling of DNA has useful characteristics for application in microarray analyses because of the large signal enhancement.

Interobserver variability for the WHO classification of pulmonary carcinoids.

Pulmonary carcinoids are neuroendocrine tumors histopathologically subclassified into typical (TC; no necrosis, <2 mitoses per 2 mm) and atypical (AC; necrosis or 2 to 10 mitoses per 2 mm). The reproducibility of lung carcinoid classification, however, has not been extensively studied and may be hampered by the presence of pyknotic apoptosis mimicking mitotic figures. Furthermore, prediction of prognosis based on histopathology varies, especially for ACs. We examined the presence of interobserver variation between 5 experienced pulmonary pathologists who reviewed 123 originally diagnosed pulmonary carcinoid cases. The tumors were subsequently redistributed over 3 groups: unanimously classified cases, consensus cases (4/5 pathologists rendered identical diagnosis), and disagreement cases (divergent diagnosis by ≥2 assessors). κ-values were calculated, and results were correlated with clinical follow-up and molecular data. When focusing on the 114/123 cases unanimously classified as pulmonary carcinoids, the interobserver agreement was only fair (κ=0.32). Of these 114 cases, 55% were unanimously classified, 25% reached consensus classification, and for 19% there was no consensus. ACs were significantly more often in the latter category (P=0.00038). The designation of TCs and ACs by ≥3 assessors was not associated with prognosis (P=0.11). However, when disagreement cases were allocated on the basis of Ki-67 proliferative index (<5%; ≥5%) or nuclear orthopedia homeobox immunostaining (+; -), correlation with prognosis improved significantly (P=0.00040 and 0.0024, respectively). In conclusion, there is a considerable interobserver variation in the histopathologic classification of lung carcinoids, in particular concerning ACs. Additional immunomarkers such as Ki-67 or orthopedia homeobox may improve classification and prediction of prognosis.

The role of conventional bronchoscopy in the workup of suspicious CT scan screen-detected pulmonary nodules.

BACKGROUND: Up to 50% of the participants in CT scan lung cancer screening trials have at least one pulmonary nodule. To date, the role of conventional bronchoscopy in the workup of suspicious screen-detected pulmonary nodules is unknown. If a bronchoscopic evaluation could be eliminated, the cost-effectiveness of a screening program could be enhanced and the potential harms of bronchoscopy avoided. METHODS: All consecutive participants with a positive result on a CT scan lung cancer screening between April 2004 and December 2008 were enrolled. The diagnostic sensitivity and negative predictive value were calculated at the level of the suspicious nodules. In 95% of the nodules, the gold standard for the outcome of the bronchoscopy was based on surgical resection specimens. RESULTS: A total of 318 suspicious lesions were evaluated by bronchoscopy in 308 participants. The mean ± SD diameter of the nodules was 14.6 ± 8.7 mm, whereas only 2.8% of nodules were > 30 mm in diameter. The sensitivity of bronchoscopy was 13.5% (95% CI, 9.0%-19.6%); the specificity, 100%; the positive predictive value, 100%; and the negative predictive value, 47.6% (95% CI, 41.8%-53.5%). Of all cancers detected, 1% were detected by bronchoscopy only and were retrospectively invisible on both low-dose CT scan and CT scan with IV contrast. CONCLUSION: Conventional white-light bronchoscopy should not be routinely recommended for patients with positive test results in a lung cancer screening program.

EGFR and KRAS mutations in lung carcinomas in the Dutch population: increased EGFR mutation frequency in malignant pleural effusion of lung adenocarcinoma.

BACKGROUND: Frequencies of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) have predominantly been determined in East Asian and North American populations, showing large differences between these populations. The aim of the present study was to determine the frequency of EGFR and KRAS mutations in NSCLC in the West European Dutch population in primary carcinomas and different metastatic locations. METHODS: EGFR (exons 19, 20 and 21) and KRAS (exons 2 and 3) mutation test results of NSCLC samples of patients in 13 hospitals were collected. The tests were performed on paraffin-embedded tissue or cytological material of primary and metastatic lung carcinomas. RESULTS: EGFR mutations were detected in 71/778 (9.1 %) tested patients; in 66/620 (10.6 %) adenocarcinomas. EGFR mutations were significantly more often detected in female than in male patients (13.4 % vs. 5.5 %, p < 0.001). KRAS mutations were found in 277 out of 832 (33.3 %) tested patients; in 244/662 (36.9 %) adenocarcinomas. A significantly increased frequency of EGFR mutations was observed in patients with malignant pleural/pericardial effusions (26.5 %; odds ratio (OR) 2.80, 95 % confidence interval (CI) 1.22-6.41), whereas the frequency of KRAS mutations was significantly decreased (18.8 %; OR 0.35, 95 % CI 0.14-0.86). CONCLUSIONS: In the investigated Dutch cohort, patients with malignant pleural/pericardial effusion of lung adenocarcinoma have an increased frequency of EGFR mutations. The overall frequency of EGFR mutations in lung adenocarcinomas in this West European population is within the frequency range of North American and South European populations, whereas KRAS mutation frequency is higher than in any population described to date.

The role of the ¹8f-fluorodeoxyglucose-positron emission tomography scan in the Nederlands Leuvens Longkanker screenings Onderzoek lung cancer screening trial.

BACKGROUND: In computed tomography lung cancer screening programs, up to 30% of all resections are futile. OBJECTIVE: To investigate whether a preoperative positron emission tomography (PET) after a conclusive or inconclusive nonsurgical workup will reduce the resection rate for benign disease in test-positive participants of a lung cancer screening program. METHODS: ¹8F-Fluorodeoxyglucose-PET scans were made in 220 test positives. Nodules were classified as positive, indeterminate, or negative based on visual comparison with background activity. Gold standard for a positive PET was the presence of cancer in the resection specimen or the detection of cancer during more than 2 years follow-up. Sensitivity, specificity, positive predictive value, and negative predictive value (NPV) were calculated at participant level and 95% confidence intervals (CIs) constructed. RESULTS: The sensitivity of PET to detect cancer was 84.2% (95% CI: 77.6-90.7%), the specificity 75.2% (95% CI: 67.1-83.3), the positive predictive value 78.9% (95% CI: 71.8-86.0), and the NPV 81.2% (95% CI: 73.6-88.8). The resection rate for benign disease was 23%, but 26% of them had a diagnosis with clinical consequences. A preoperative PET after an inconclusive nonsurgical workup reduced the resection rate for benign lesions by 11 to 15%, at the expense of missing 12 to 18% lung cancer cases. A preoperative PET after a conclusive nonsurgical workup reduced the resection rate by 78% at the expense of missing 3% lung cancer cases. CONCLUSION: A preoperative PET scan in participants with an inconclusive nonsurgical workup is not recommended because of the very low NPV, but after a conclusive nonsurgical workup, the resection rate for benign disease can be decreased by 72%.

The clinical value of lymphatic micrometastases in patients with non-small cell lung cancer.

INTRODUCTION: In early stage non-small cell lung cancer (NSCLC), presence of lymphatic micrometastases and isolated tumor cells, primarily detected by immunohistochemistry, is suggested to be a prognostic factor. However, there is no consensus whether immunohistochemistry should be used routinely in lymph node assessment.The goal of our study was to determine whether recurrent disease is associated with the presence of lymphatic micrometastases and/or isolated tumor cells, at the time of the lung resection. METHODS: We retrospectively analyzed the prevalence of lymphatic micrometastases and/or isolated tumor cells in two groups of patients, who underwent a curative resection for early stage NSCLC. Group I had a follow-up of 5 years without recurrent disease. Group II consisted of a matched group of patients with recurrent disease. Patients were originally classified as having negative mediastinal lymph nodes.All lymph nodes obtained by mediastinoscopy and thoracotomy were re-examined by serial sectioning and immunohistochemistry. RESULTS: Micrometastases and/or isolated tumor cells were found in one of 16 patients in group I, which was significantly different from six of 16 patients in group II. (Fisher exact test, 4.6; p, 0.04; risk ratio, 2.4).Serial sectioning and immunohistochemistry did not change N-stage for the single patient in group I, in contrast to all six patients in group II. CONCLUSION: Presence of lymphatic micrometastases and/or isolated tumor cells is associated with distant recurrence in patients with early stage NSCLC. We recommend the routine use of serial sectioning and immunohistochemistry in lymph node assessment to improve the accuracy of staging.

A multicenter phase II study of erlotinib and sorafenib in chemotherapy-naive patients with advanced non-small cell lung cancer.

PURPOSE: This multicenter, phase II study evaluates the efficacy and safety of erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, plus sorafenib, a multityrosine kinase inhibitor against vascular endothelial growth factor receptors, in patients with previously untreated advanced non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Chemotherapy-naïve patients with stage IIIB/IV NSCLC received erlotinib (150 mg once a day) and sorafenib (400 mg twice a day) until disease progression or unacceptable toxicity. The primary end point was the rate of nonprogression at 6 weeks. Secondary end points included objective response rate (ORR), time to progression, overall survival, and adverse events. Exploratory end points included pretreatment EGFR and KRAS mutation status, pharmacokinetics, and cytochrome P450 polymorphisms. RESULTS: Fifty patients initiated therapy. The nonprogression rate at 6 weeks was 74%: 12 (24%) partial response and 25 (50%) stable disease. Ultimately, the ORR was 28%. Median time to progression was 5.0 months [95% confidence interval (95% CI), 3.2-6.8 months]. Median overall survival was 10.9 months (95% CI, 3.8-18.1 months). Grade 3/4 adverse events included fatigue (16%), hand-foot skin reaction (16%), rash (16%), diarrhea (14%), and hypophosphatemia (42%). There was one treatment-related fatal pulmonary hemorrhage. Patients with wild-type EGFR had a higher ORR (19%) than previously reported for single-agent erlotinib/sorafenib. Erlotinib levels were lowered. This was associated with CYP3A4 polymorphism and was possibly due to sorafenib. CONCLUSION: Despite a possible drug interaction, sorafenib plus erlotinib has promising clinical activity in patients with stage IIIB/IV NSCLC and has an acceptable safety profile. Further evaluation of this combination as potential salvage therapy in EGFR mutation-negative patients and the possible drug interaction is warranted.

Circulating DNA is a non-invasive prognostic factor for survival in non-small cell lung cancer.

INTRODUCTION: Circulating plasma DNA is present in a considerably higher concentration in lung cancer patients than in controls. Conflicting data are reported about circulating DNA as a prognostic factor. The aim of this study was to prospectively analyse the relationship of circulating plasma DNA with overall survival (OS) of previously untreated non-small cell lung cancer (NSCLC) patients. METHODS: 46 untreated NSCLC patients and 21 controls with a follow-up time of 6.5 years were analyzed. Quantification of baseline circulating plasma DNA was performed by a real-time quantitative polymerase chain reaction (qPCR) targeting the human beta-globin gene. Survival analysis was performed using the Kaplan-Meier method and compared with a Cox-regression analysis. RESULTS: The median DNA concentration of the patients who died (87%) was significantly higher compared to the patients that survived at the end of follow-up (55ng/ml versus 23ng/ml, p=0.02). In patients with higher DNA concentration overall survival was significantly worse. In this study no relation of DNA concentration with tumour characteristics, age, gender or pulmonary inflammatory conditions was found. CONCLUSION: In this study a high circulating plasma DNA concentration at time of diagnosis in NSCLC patients was a prognostic factor for poorer survival. Circulating DNA may be used as a non-invasive biomarker to refine the prognostic profile in NSCLC patients.

Acetylcholine receptor pathway and lung cancer.

Genome-wide association studies revealed chromosome regions 15q24-25 were associated with a higher risk for development of lung cancer. The 15q24-25 region encompasses the nicotinic acetylcholine receptor subunit genes (nAchR alpha3, alpha5, and beta4) that play a role in nicotine addiction. This review reports information of the acetylcholine receptor and lung cancer. In patients diagnosed with smoking-related lung cancer and who continue smoking, a negative correlation with lung cancer survival has been shown. The reduced treatment efficacy may well be explained by the pluriform effect of nicotine on tumor cell proliferation, apoptosis, epithelialmesenchymal transition, proinvasive, and angiogenic effects, which are reenforced by an autocrine/paracrine loop. Overall, there is no evidence that nicotine itself induces cancer, but nicotine promotes in vivo the growth of cancer cells and the proliferation of endothelial cells. This suggests that nicotine after initiation may contribute to the progression phase of cancer development. Continuation of smoking after lung cancer has been diagnosed should be discouraged because smoking may not only support tumor growth but also interferes with treatment.

Forced lordosis on the thoracolumbar junction can correct coronal plane deformity in adolescents with double major curve pattern idiopathic scoliosis.

STUDY DESIGN: A prospective radiographic study was conducted. OBJECTIVE: To support our hypothesis that correction of the scoliosis may benefit from a lordotic fulcrum force in the sagittal plane on the thoracolumbar spine. SUMMARY OF BACKGROUND DATA: Adolescent idiopathic scoliosis is an important spinal deformity. Correction can be achieved with limited options by current bracing techniques. Lateral bending radiographs are used to assess flexibility and predict treatment outcome. The corrective potential of a lordotic fulcrum force in the sagittal plane has not been addressed. METHODS: Anterioposterior spine radiographs of patients with a double major curve pattern scoliosis were obtained in 2 groups of patients. In group A radiographs in 3 positions: standing, and supine with and without fulcrum (n = 12), and group B radiographs in 2 positions (n = 28): standing, and supine with lordotic fulcrum. Cobb angles were determined and evaluated statistically. The sagittal contour of the thoracolumbar junction in standing position was measured. RESULTS: In group A with the patients lying supine a correction of the Cobb angle was obtained at the thoracic level of 15.4% and the lumbar level of 27.5% (P < 0.001). Adding in supine position a lordotic fulcrum on the thoracolumbar junction resulted in a coupled further correction at the thoracic level of 15.7% and lumbar 18.1% (P < 0.001). Comparing in group A the thoracic and lumbar curvatures in standing position with that on a lordotic fulcrum in supine position revealed a total reduction of 31% and 45.6%, respectively. For the independent group B this reduction in 1 step is 38% and 44.4%, respectively. CONCLUSION: Scoliotic deformities are significantly reduced in supine position by a lordotic fulcrum force on the thoracolumbar junction. These findings may have consequences on bracing techniques.

Immunohistochemical markers associated with brain metastases in patients with nonsmall cell lung carcinoma.

BACKGROUND: To the authors' knowledge, there are no reliable markers able to identify patients with nonsmall cell lung cancer (NSCLC) that will develop metastases to the brain. The authors investigated associations between immunohistochemical markers and the development of brain metastases in patients with NSCLC. METHODS: This was a hospital-based, case-control study of patients who were newly diagnosed with NSCLC between 1989 and 2003, developed brain metastases, and had pathology material available from both the primary NSCLC and the brain metastases. These patients were compared with a control group of patients who had NSCLC and no evidence of brain metastases. NSCLC was examined for expression levels of Ki-67, caspase-3, vascular endothelial growth factor A (VEGF-A), VEGF-C, E-cadherin, and epidermal growth factor receptor (EGFR) in 54 surgical pathology specimens using immunohistochemistry, and associations were evaluated between those markers and the development of brain metastases. RESULTS: Brain metastases developed after a median of 12.5 months (range, 1.7-89.4 months) after the diagnosis of NSCLC. A significantly increased risk of developing brain metastases was associated with patients with NSCLC who had primary tumors with high Ki-67 levels (adjusted odds ratio [OR] of 12.2; 95% confidence interval [95% CI], 2.4-70.4 [P < .001]), low caspase-3 expression (adjusted OR of 43; 95% CI, 5.3 to >100 [P < .001]), high VEGF-C expression (adjusted OR of 14.6; 95% CI, 2.0 to >100 [P < .001]), and low E-cadherin (adjusted OR of 3.6; 95% CI, 0.9-16.4 [P = .05]). No significant risk was associated with VEGF-A or EGFR expression. High Ki-67 expression also was associated with a shorter overall survival (P = .04). CONCLUSIONS: The results of the current study indicated that patients with NSCLC who had high Ki-67 expression, low caspase-3 expression, high VEGF-C expression, and low E-cadherin expression in their tumors may benefit from close surveillance because they may have an increased risk of developing brain metastases.

Cytoglobin, the newest member of the globin family, functions as a tumor suppressor gene.

Cytoglobin (CYGB) is a recently discovered vertebrate globin distantly related to myoglobin with unknown function. CYGB is assigned to chromosomal region 17q25, which is frequently lost in multiple malignancies. Previous studies failed to detect evidence for mutations in the CYGB gene. Recent studies provided preliminary evidence for increased methylation of the gene in lung cancer. Our study was aimed at investigating the role of CYGB as a tumor suppressor gene. By nested methylation-specific DNA sequencing analysis of lung and breast cancer cell lines and bronchial and mammary epithelial cell lines, we identified that methylation of a 110-bp CpG-rich segment of the CYGB promoter was correlated with gene silencing. We specifically targeted this sequence and developed a quantitative methylation-specific PCR assay, suitable for high-throughput analysis. We showed that the tumor specificity of CYGB methylation in discriminating patients with and without lung cancer, using biopsies and sputum samples. We further showed the tumor specificity of this assay with multiple other epithelial and hematologic malignancies. To show tumor suppressor activity of CYGB, we performed the following: (a) RNA interference-mediated knockdown of CYGB gene on colony formation in a CYGB expression-positive lung cancer cell line, resulting in increased colony formation; (b) enforced gene expression in CYGB expression-negative lung and breast cancer cell lines, reducing colony formation; and (c) identification of potential proximate targets down-stream of the CYGB genes. Our data constitute the first direct functional evidence for CYGB, the newest member of the globin family, as a tumor suppressor gene.

Risk factors for non-sentinel lymph node metastases in patients with breast cancer. The outcome of a multi-institutional study.

BACKGROUND: In this multi-institutional prospective study, we evaluated whether we could identify risk factors predictive for non-sentinel lymph node (non-SN) metastases in breast cancer patients with a positive sentinel lymph node (SN). METHODS: In this multi-institutional study, 541 eligible breast cancer patients were included prospectively. RESULTS: The occurrence of non-SN metastases was related to the size of the SN metastasis (P = .02), primary tumor size (P = .001), and lymphovascular invasion (P = .07). The adjusted odds ratio was 3.1 for SN micro-metastasis compared with SN isolated tumor cells, 4.0 for SN macro-metastasis versus SN isolated tumor cells, 3.1 for tumor size (>3.0 cm compared with 3.0 cm, and with vessel invasion. CONCLUSION: We identified three predictive factors for non-SN metastases in breast cancer patients with a positive SN: size of the SN metastasis; primary tumor size; and vessel invasion. We were not able to identify a specific group of patients with a positive SN in whom the risk for non-SN metastases was less than 5%.